The overall objective of this renewal application is the development of DNA-mediated electrochemistry as a new technology for sensing DNA-binding proteins and nucleic acids. DNA-mediated electrochemistry is remarkably sensitive in detecting perturbations in the DNA base pairs and hence can be used as a sensor for changes in base stacking that arise with base mismatches or protein binding. Electrical sensors for mutation detection and for base flipping proteins that bind to DNA have been developed using this chemistry. In the program proposed, the scope of this chemistry will be expanded to develop new tools to monitor proteins that bind to DNA. Specifically, DNA-modified gold and graphite electrodes will be used to probe interactions and reactions of proteins on the duplex. DNA-bound redox potentials of redox-active transcriptional activators will be determined. For proteins that lack redox cofactors, covalent redox probes on the DNA duplex will be used as the reporter, and proteins with different DNA binding motifs will be examined. We can also monitor reactions of photolyase and PvuII on DNA to probe the DNA conformational changes that occur during reaction. Thus DNA electrochemistry offers a new tool to probe how proteins bind and react on DNA. DNA-mediated electrochemistry also offers high sensitivity and the ability to multiplex. By systematically reducing the DNA-modified electrode size down to 100 nm, and utilizing electrocatalysis for signal amplification, we will construct sensitive electrical sensors for proteins that bind to DNA and activate transcription. Microelectrodes offer faster kinetics, as well as sensitivity in monitoring protein binding. Multiplexed arrays of DNA-modified electrodes will be fabricated first on the macroscale for optimization. Initial designs will be used to test for methylases that bind different target sequences. A multiplexed assay to test for DNA-binding proteins will then be constructed based upon a competition array with the methylases. Assays for sequence-specific inhibitors of protein binding will also be developed. Using similar approaches, we will examine the scope and limits of RNA detection electrochemically, how DNA-mediated detection can be made robust and quantitative in detecting mRNAs at low concentrations. We will also combine these assays to develop arrays where mRNAs and the transcription factors that activate their synthesis can be detected simultaneously. These sensors offer the potential for a completely new technology to monitor DNA-binding proteins and mRNAs in a label-free format. These tools could provide a sensitive new diagnostic to detect both protein and RNA markers for cancer and other disease states. PUBLIC HEALTHE RELEVANCE This project involves the construction of new electrical sensors for RNA and proteins that bind to DNA. These sensors will serve as completely new diagnostic tools to detect the proteins and RNAs from cells and tissues that are associated with different disease states. These sensors could represent a new technology for the early diagnosis of cancer and other diseases.